Research Article
Chemical Constituents and Cytotoxic Activity of Polyalthia cauliflora var. cauliflora

N.A. Ghani, N. Ahmat, N.H. Ismail, I. Zakaria and N.K.N.A. Zawawi

Research Journal of Medicinal Plants, 2012, 6(1), 74-82.

Abstract

Phytochemical investigation of the stem bark of Polyalthia cauliflora var. cauliflora has led to the isolation of two chalcones and an alkaloid; 2’, 4’-dihydroxy-3’-methoxychalcone (1), 2’, 4’-dihydroxychalcone (2) and liriodenine (3). 2’, 4’-dihydroxy-3’-methoxychalcone (1) n was found to be cytotoxic against HL-60, MCF-7 and HeLa cancer cell lines with IC50 value of 12.2, 5.1 and 12.5 μg mL-1, respectively. 2’, 4’-dihydroxy-3’-methoxychalcone may prove to be useful in cancer treatment and prevention. The aims of this study were to extract, fractionate and isolate the chemical constituents from the stem bark of Polyalthia cauliflora var. cauliflora. Various chromatographic techniques including glass column chromatography, preparative thin layer chromatography and centrifugal chromatography had been used in this study. The isolated compounds were elucidated using several spectroscopic techniques including 1H and 13C NMR, IR, UV-Vis, mass spectroscopy and comparison with literature. Potential and sufficient amount of compounds were subjected to cytotoxic activity against HL-60, MCF-7 and HeLa cancer cell lines.

ASCI-ID: 82-172

Mabberley, 1987). The genus Polyalthia (Annonaceae) consists of about 120 species of shrubs and trees. This genus is widely distributed in tropical and subtropical (Connolly et al., 1996). Alkaloid, terpenes, benzopyrans and flavonoids have been isolated from Polyalthia species (Abu Zarga and Shamma, 1982; Gonzalez et al., 1995; Islam et al., 2001; Kijjoa et al., 1990). Previous chemical investigation on Polyalthia beccarii (Hooker, 1875) and Polyalthia cauliflora Jossang et al. (1982) reported several bisaporphine-typed of alkaloids.

Polyalthia has been used as folk medicine in many tropical countries. In India, Polyalthia plants has been used as bitter tonic, abortifacient, febrifuge, a cure for scorpion stings, high blood pressure and as respiratory stimulant (Padmaa and Khosa, 2009). In addition, seeds and bark of P. longifolia are used as febrifuge in the Balasore district of Orissa (Raghunathan and Mitra, 1982). In Thai, a water decoction of the root of P. evecta has been used by the North-Eastern natives as a galactagogue (Kanokmedhakul et al., 1998). Furthermore, P. lateriflora was used to treat skin infection (Wiart, 2000) and the alkaloids isolated from its stem bark have been reported for antibacterial and antifungal activities (Hasan et al., 1988). Polyalthia cauliflora was used by the Kelabit community in Bario, Sarawak for birth control (Fasihuddin et al., 1995).

Chalcone, a type of compound isolated from P. cauliflora var. cauliflora is an aromatic ketone which fordms the central core for a variety of important biological compounds (Mandge et al., 2007). Chalcones and their derivatives are also medicinally important as they were reported to display various biological activities such as antioxidant, antimicrobial, antibacterial, antifungal, antitumor and anti-inflamantory (Alam et al., 2004; Alam and Mostahar, 2005; Azad et al., 2007; Lotulung et al., 2008; Oyedapo et al., 2008). Some chalcones showed the ability to block voltage-dependent potassium channels (Yarishkin et al., 2008). Chalcone is an intermediate in the biosynthesis of flavonoid which substance is widespread in plants with an array of biological activities (Martens and Mithofer, 2005; Mostahar et al., 2007).

The intention of this study was to isolate the chemical constituents from the methanolic extract of the stem bark of P. cauliflora var. cauliflora. The isolation and purification of the non alkaloid fraction has led to the isolation of two chalcone; 2’, 4’-dihydroxy-3’-methoxychalcone and 2’, 4’-dihydroxychalcone while the isolation of the alkaloidal fraction yielded an alkaloid; liriodenine.

MATERIALS AND METHODS

General experimental procedures: The 1H-NMR and 13C-NMR were recorded in chloroform-D on Bruker 300 Ultrashield NMR spectrometer measured at 300 and 75 MHZ. Chemical shifts are reported in ppm and the coupling constants are given in Hz. Melting point was taken on a hot stage Gallen Kamp melting point apparatus with microscope and was uncorrected. The Infrared (IR) was recorded on the Perkin Elmer spectrum one FT-IR spectrometer. The Ultraviolet (UV) spectra were recorded on Shimadzu UV-Vis 160i. The mass spectra were measured on Perkin Elmer Clarus 600T spectrometer 70 eV. Glass column used silica gel 60 230-400 mesh ASTM (Merck 1.09385), centrifugal thin layer chromatography used silica gel 60 PF254 (Merck catalog number: 1.07749). Aluminum supported silica gel 60 F254 was used for analytical thin layer chromatography while glass supported silica gel 60 F254 was used for preparative thin layer chromatography.

Extraction and isolation: The stem bark of P. cauliflora var. cauliflora was collected from Kuala Keniam, Pahang and a voucher specimen (UiTM64/2008) was deposited at the Faculty of Applied Sciences Herbarium, Universiti Teknologi Mara (UiTM), Malaysia. The stem bark (350 g) was air-dried, ground and macerated in methanol for several times. The extract was filtered and concentrated under reduced pressure. The crude extract (16.25 g) was subjected to acid-base extraction yielding a non-alkaloidal fraction that was subjected to silica gel column chromatography and eluted with hexane 100% and increasing polarity with DCM, EA and MeOH to afford 52 fractions. Fractions with similar profile on analytical TLC were pooled together to yield 12 sub fractions. Sub fraction 10 was subjected to purification by multiple development of Preparative Thin Layer Chromatography (PTLC) using hexane: ethyl acetate 9:1 system to afford compound 1. Re-column chromatography and further purification using multiple development-preparative thin layer chromatography [Hex:DCM (4:6)] of subfraction 12 gave compound 2. as yellow oil. The alkaloidal crude (4.9374 g) was subjected to fractionation using Vacuum Liquid Chromatography (VLC) with various composition of solvent system [Hex: DCM (5:5, 0:10) and DCM: MeOH (9.5:0.5, 9:1, 8.5:1.5, 8:2,5:5, 0:10)] to yield 11 fractions. Fraction 3 was subjected to centrifugal chromatography to afford compound 3.

2’, 4’-dihydroxy-3’-methoxychalcone (1): Yellow oil MS m/z: 270 C16H14O4. UV λmax nm MeOH: 201, 245, 253, 279, 290, 311, 360, 370. IR cm-1: 3434, 2987, 1638, 1263.

2’,4’-dihydroxychalcone (2): Yellow needle crystal MS m/z: 241 C15H12O3. UV λ max nm MeOH: 202, 208, 246, 277, 294, 313, 317, 401. IR cm-1: 3433, 1639, 1267.

Liriodenine (3): Yellow needle crystal MS m/z: 275(M+); UV (EtOH):215, 246, 268, 395, 412 nm; IR (KBr) cm-1:3054, 2685, 1726, 1421, 1265.

Cytotoxic assay: The cytotoxicity assay was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay (Khorshid et al., 2011). Cells were seeded in 96-well microplate at 3x105 cells/mL, incubated at 37°C in 5% CO2 and treated with sample at 2xMFC in Serum Free Medium (SFM) for 240 and 480 min. The culture medium was aspirated, replaced with 0.5 μg mL-1 MTT solutions and incubated for 30 min in a CO2 incubator. The solution was aspirated and added with 1,000 μL DMSO to dissolve the formazan crystals. After 30 min of rotary agitation, the absorbance of the solution was measured at 570 nm using Genesis10 UV-Vis spectrophotometer (Thermo Spectronic, NY, USA). The viable cell number was calculated from the standard curve of cell number by plotting a scattergram of the absorbance value against the known number of cells. IC50 values represent the compound’s concentration that reduced the mean absorbance at 570 nm to 50% of those in the untreated control wells.

RESULTS AND DISCUSSION

Two chalcones and an alkaloid were isolated from the stem bark of P. cauliflora var. cauliflora. Both chalcones 1 and 2 have a phenolic OH substituted at position C-2’ of A ring and a monosubstituted ring B while the alkaloid is an oxoaporphine type of alkaloid (Fig. 1).

Chalcone 1 (13.4 mg) was isolated as yellow oil. The UV spectrum showed maxima absorption at 201, 245, 253, 279, 290, 311, 344, 360 and 370 nm which are typical of chalcone moiety. The IR spectrum indicated the presence of hydroxyl group at 3434 cm-1, C-H aromatic at 2987 cm-1, C = O at 1638 cm-1 and C-O at 1263 cm-1. The mass spectrum showed a molecular ion peak at 270 m/z corresponding to the molecular formula C16H14O4.

Fig. 1: Chalcones and oxoaporphine isolated from P. cauliflora var. cauliflora

1H NMR spectrum of 1 showed a mutual pair of doublet at δ 7.92 and δ 7.56 (J = 15.6 Hz) suggesting the presence of trans olefin protons H-β and H-α, respectively. A pair of doublets observed at δ 6.59 and δ 7.64 (J = 9.0 Hz) are assignable to ortho-coupled protons at H-5’ and H-6’, respectively while two multiples at δ 7.46 and δ 7.68 integrated as three and two protons, respectively are characteristic of a monosubstituted ring B. These sets of signal gave clear indication that this compound is a chalcone. A sharp peak at a very downfield region at δ 13.57 is assignable to a phenolic OH chelated to the oxygen of the carbonyl group next to it (Zakaria et al., 2010). A signal of methoxy group could be observed at δ 4.05.

The 13C NMR spectrum exhibited 14 signals representing 16 carbons with a carbonyl carbon signal at a very down field region at δ 192.6. Three oxyaryl carbons can be observed at δ 134.3, 155.3 and 157.8 corresponding to C-3’, C-4’ and C-2’, respectively while two quaternary aromatic carbons gave signals at δ 115.0 (C-1’) and 134.7 (C-1). Signals at δ 120.2 and 144.7 belong to two olefin sp2 carbons present in the chalcone nucleus while signals for six aromatic C-H can be observed at δ 106.5 (C-5’), 129.3 (C-6’), 128.6 (C-2/C-6), 129.0 (C-3/C-5) and 130.8 (C-4). Finally a methoxy signal was detected at δ 60.8. These 13C NMR together with 1H NMR data confirmed that this compound is a chalcone with a monosubstituted ring B and a methoxy group located at ring A. Comparison with literature showed close resemblance of this compound with those reported for 2’,4’-dihydroxy-3’-methoxychalcone (Svetaz et al., 2004).

Chalcone 2 (14.5 mg) was isolated as a yellow needle crystal. The UV spectrum showed the absorption maxima at 202, 208, 246, 277, 294, 313, 317 and 401 nm which are typical of chalcone moiety. The IR spectrum exhibited the presence of OH, C = O and C-O groups at 3433 cm-1, 1639 cm-1 and 1267 cm-1 respectively. The mass spectral showed a molecular ion peak at m/z 240 suggesting the molecular formula of C15H12O3.

The 1H NMR spectrum (Table 1) exhibited a very downfield signal (singlet) at δ 13.41 indicative of a chelated hydroxyl group at position C-2’.

Table 1: 1H and 13C NMR of compound 1 and 2

Two multiples observed in the aromatic region at δ 7.45 and δ 7.68 are characteristic of monosubstituted ring B and a pair of doublets at δ 7.59 and δ 7.91 (J = 15.6 Hz) are attributed to α and β protons in trans position. An ABD system could be observed in this structure with the presence of signals at δ 7.85 (d, J = 9.3 Hz), δ 6.49 (dd, J = 9.3, 2.1 Hz) and δ 6.46 (d, J = 2.1 Hz).

The 13C NMR spectrum of 1 showed similar pattern of signals as in compound 2 assignable to chalcone skeleton. An additional signal at δ 103.8 in the spectrum suggests an unsubstitution of C-3’ (methine sp2 carbon). Based on the spectral data and comparison with literature, compound 2 was characterized as 2’, 4’-dihydroxychalcone previously reported from the aerial parts of Galenia africana (Aizoaceae) (Vries et al., 2005). This is the first occurrence of this compound in Polyalthia genus.

Compound 3 (1.3 mg) was obtained as yellow needles exhibiting an M+ at m/z 275, corresponding to molecular weight C17H9O3N. The UV spectrum showed absorbance bands at 215, 246, 268, 395 and 412 nm suggesting an aporphine moiety. The IR spectrum indicated the presence of C-H aromatic at 3054 cm-1, conjugated C = N group at 2685 cm-1, C = O group at 1726 cm-1 and C-O group at 1265 cm-1.

The 1H NMR spectrum of 3 (Table 2) showed signals of seven aromatic and a methylenedioxy protons. A mutual-coupled protons were observed at δ 6.40 and 7.79 and 8.90 (d, J = 5.1 Hz). Two singlets at δ 6.40 and δ 7.16 were attributed to protons of methylenedioxy and aromatic H-3 respectively. The rest of the signals (δ 6.59, 7.60, 7.64 and 7.92) belong to four methine sp2 protons in ring D.

The J-mod 13C NMR spectrum of 3 showed the presence of 17 carbons with eight quaternary carbons. One carbonyl carbon appeared at a very downfield region (δ 182.4) while two oxyaryl carbons gave signals at δ 147.9 and 151.7. Signals for six quaternary carbons could be observed at δ 108.2 (C-1b), 131.3 (C-11a), 132.9 (C-7a), 135.7 (C-1a), 145.4 (C-3a) and 146.0 (C-6a) while signal of a methylenedioxy carbon could be seen at δ 102.4. These spectral data suggested compound 3 as an oxoaporphine alkaloid and comparison with the literature confirmed it to be liriodenine (Lavault et al., 1981).

Table 2: 1H and 13C NMR of compound 3

Table 3: IC50 value of compound 1 tested against HeLa, HL-60 and MCF-7 cancer cell lines
*Range of activity (inhibition): <5: Very strong, 5-10: Strong, 10-20: Moderate, 20-100: Weak, >100: Not active (Wibowo et al., 2011)

Compound 3 was reported to display good activity against antifungal and cytotoxic (brine shrimp bioassay) (Rahman et al., 2005), anticancer against lung cancer cells (Chang et al., 2004) and antiparasitic activities (Fernandez et al., 2010).

2’, 4’-dihydroxy-3’-methoxychalcone (1) was assayed against three cancer cell lines which were HeLa (human servical cancer), HL-60 (human T-promyelocytic leukemia) and MCF-7 (human breast adenocarcinoma cancer) using MTT assay. H2O2 was used as standard. Table 3 showed that 2’,4’-dihydroxy-3’-methoxychalcone (1) exhibited in vitro cytotoxic effect against the three human cancer cell lines. It inhibited strongly the growth of HL-60 cell line with IC50 value of 5.1 μg mL-1 and moderately HeLa and MCF-7 cell lines with IC50 of 12.2 and 12.5 μg mL-1, respectively.

Apoptosis of tumor cells can be triggered by a diversity of extracellular and intracellular factors including cytokines, tumor suppressor genes, oncogenes, radiation and anticancer drugs. The growth inhibition by 2’, 4’-dihydroxy-3’-methoxychalcone (1) in the human cell lines may be due to apoptosis as nuclear condensation and fragmentation and DNA ladder formation was observed in all of the cell lines. Like any other chalcones, compound 1 chemically consists of two aromatic rings joined by three carbons, α-β-unsaturated carbonyl system. The reactivity of the carbonyl group and hydroxyl groups at position C-2’ and C-4’ in 1 is considered to be implicated in the internalization into the cell which in turn leads to interaction with the signal transduction molecules and the proteins involved in mitochondria permeability transition.

Compound 1 has been reported to display an activity against Phomopsis longiculla Hobbs CE117 for antifungal assay (Nowakowska, 2007) 2’, 4’-dihydroxychalcone (2) also was reported to possess diverse pharmacological activities such as antiprotozoal, antifungal, antiMRSA, antiinflamatory, interleukin-1 and tyrosinase inhibitory, phytoesterogenic, antipyretic, analgesic, antioxidant and anticancer activities (Anto et al., 1995; Edenharder and Tang, 1997; Lopez et al., 2001; Nerya et al., 2004; Uchiumi et al., 2003; Wu et al., 2003). Thus, compound 2 is expected to display good cytotoxic effect as shown in 1 given that both compounds have hydroxyl group as substituent.

CONCLUSION

In summary, the phytochemical study of P. cauliflora var. cauliflora has led to the isolation of two chalcone; 2’,4’-dihydroxy-3’-methoxychalcone (1) and 2’,4’-dihydroxychalcone (2) and one alkaloid; liriodenine (3). Polyalthia of Annonaceae is known for its high content of alkaloid but we report here the isolation of chalcones as well. The results from MTT assay showed that chalcone 1, exhibited antiproliferative activity against human leukemia HL60 cells, human cervical cancer HeLa cells and human breast adenocarcinoma MCF-7 cells. Thus, 1 had been shown to induce apoptosis in leukemia cell lines.

**ad2**

ACKNOWLEDGMENTS

This study was supported by grant from FRGS (No: 600-RMI/ST/FRGS/5/3/Fst (32/2009)) and scholarship of one of the author was financed by National Science Fellowship (NSF) from Ministry of Science, Technology and Innovation, Malaysia (MOSTI).

" class="btn btn-success" target="_blank">View Fulltext

Similar Articles


Cytotoxicity and Genotoxicity of Tridax procumbens L. in Allium cepa

Research Journal of Medicinal Plants, 2016, 10(1), 120-126.

Cited By


Design, synthesis and QSAR study of 2′-hydroxy-4′-alkoxy chalcone derivatives that exert cytotoxic activity by the mitochondrial apoptotic pathway

Bioorganic & Medicinal Chemistry, 2018, (), . DOI: 10.1016/j.bmc.2018.10.045

Mechanism Study of Green Synthesis and Antibacterial Attribute of <i>Polyalthia longifolia</i> Based Gold Nanoparticles

Journal of Nano Research, 2022, 75(), 1. DOI: 10.4028/p-k39913

Assessing the pollution effect of cement dust emission on the soil quality around Ewekoro cement factory, southwestern Nigeria

Environmental Forensics, 2022, (), 1. DOI: 10.1080/15275922.2022.2125120

Evaluation of biological activity and in silico molecular docking studies of Acanthus ilicifolius leaf extract against four multidrug-resistant bacteria

Informatics in Medicine Unlocked, 2022, 33(), 101092. DOI: 10.1016/j.imu.2022.101092

Comparison of pharmacokinetics of marbofloxacin after subcutaneous administration of various multiple-dose regimens to water buffalo calves (Bubalus bubalis)

American Journal of Veterinary Research, 2014, 75(12), 1049. DOI: 10.2460/ajvr.75.12.1049

Invited Review: Using whole cottonseed and cotton harvest residue in southeastern US beef cattle diets: Quality, intake, and changes in feed characteristics

Applied Animal Science, 2022, 38(5), 447. DOI: 10.15232/aas.2022-02301

Eichhornia crassipes expansion detection using geospatial techniques: Lake Dambal, Oromia, Ethiopia

Environmental Challenges, 2022, 9(), 100616. DOI: 10.1016/j.envc.2022.100616

Bacterial Milk Quality along the Value Chain in Smallholder Dairy Production

The Scientific World Journal, 2022, 2022(), 1. DOI: 10.1155/2022/7967569

Non-food bioactive forest product liriodenine: Sources, chemistry, and bioactivities

Industrial Crops and Products, 2022, 187(), 115447. DOI: 10.1016/j.indcrop.2022.115447

An assessment, prospects, and obstacles of industrially important medicinal crop Indian Senna (Cassia angustifolia Vahl.): A review

Industrial Crops and Products, 2022, 187(), 115472. DOI: 10.1016/j.indcrop.2022.115472

Secondary metabolic profile of rose-scented geranium: A tool for characterization, distinction and quality control of Indian genotypes

Industrial Crops and Products, 2022, 187(), 115487. DOI: 10.1016/j.indcrop.2022.115487

Extraction, characterization and epoxidation of Cassia fistula (Indian laburnum) seed oil: A bio-based material

Industrial Crops and Products, 2022, 187(), 115496. DOI: 10.1016/j.indcrop.2022.115496

Undescribed meleagrin alkaloids from the endophytic fungus Penicillium commune

Phytochemistry, 2022, (), 113441. DOI: 10.1016/j.phytochem.2022.113441

Biodiesel production from swine manure via housefly larvae (Musca domestica L.)

Renewable Energy, 2014, 66(), 222. DOI: 10.1016/j.renene.2013.11.076

Effects of sodium and magnesium supplement on lipid production and wastewater treatment by Rhodosporidium toruloides

Renewable Energy, 2022, 199(), 919. DOI: 10.1016/j.renene.2022.09.043